Project Summary To accomplish their diverse maintenance and protective roles, microglia must be extremely plastic, dynamically sensing and responding to specific local challenges throughout the brain. Recent results have called into question whether state-of-the-art models of microglial biology accurately capture the rich interactive environment encountered by human microglia in the living brain. To understand microglial biology, we need new robust and experimentally feasible technologies to systematically reveal the dynamic nature of these cells. Herein we aim to validate a new chimeric platform for exploring IPSC-derived microglial cells that we call the XMG platform. Initial transcriptional and histochemical analyses strongly support the notion that our that XMG cells have highly similar genetic profiles and activity to endogenous human microglia. Specific Aim 1 focuses on comparing the transcriptional landscape of our XMG system to endogenous microglia cells from mouse and human patients. Specific Aim 2 will rely upon cutting-edge in vivo multiphoton imaging experiments to establish the kinetic calcium activity and anatomical remodeling of XMGs in the context an acute, local laser-induced insult. Specific Aim 3 will create a novel XMG reporter system to image and isolate activated microglia for single-cell transcriptomic analyses. Additional efforts will focus on employing this system in the context of -amyloid plaque pathology (a hallmark of Alzheimer?s disease) and seek to identify any sex differences of such responses. Successful completion of these aims will result in multiple technological deliverables, such as novel microglial model systems, extensive genetic analysis of our XMG system in response to brain insults, publicly available transcriptomic web portals for easy public access, and novel human iPSC lines for easy dissemination to the field. Lastly, developing a robust and easily-manipulated technological platform to study human microglial cells in vivo will represent a quantum leap for research on human microglial biology.